The present invention relates to the use of the E region of the transcription factor DP-3 as a target for novel assays and its use as a nuclear localisation signal.
The orderly progress of cells through the cell cycle involves a number of control points which assess the status of the intracellular and extracellular environment. A major control point, occurring as cells enter S phase, involves the cellular transcription factor E2F, a molecular implicated in the regulation of S phase gene expression (Nevins, 1992; La Thangue, 1994; Mxc3xcller, 1995; Weinberg, 1995). An important for E2F in early cell cycle control is suggested by the nature of the proteins which influence its transcriptional activity. for example, members of the group of pocket proteins, exemplified by the retinoblastoma tumour suppressor gene product (pRb), repress the transcriptional activity of E2F (Hiebert et al., 1992: Zamanian and La Thangue, 1992; 1993; Schwarz et al., 1993; Wolf et al., 1995). The ability to repress E2F correlates with the capacity of pRb, or its relatives, to negatively regulate early cell cycle progression (Hiebert et al., 1992; Zamanian and Le Thangue, 1992; Hinds et al., 1992; Zhe et al., 1995; 1995a). Furthermore, growth arrest caused by high level expression of pRb can be overcome by increasing the level of E2F (Zhu et al., 1993), implying than E2F is a principal physiological target through which pRb exerts its effects on the cell cycle. Another groups of molecules which regulate cell cycle transitions, the cyclins and their associated catalytic regulatory subunits, also interact with and control the activity of E2P (Bandara et al., 1991; Lees et al., 1992; Zhu et al., 1995b). Cyclins A. E. and D together with an appropriate catalytic subunit can influence the biological activity of pocket proteins (Hinds et al., 1992; Dowdy et al., 1993; Ewen et al., Sherr, 1993), and direct phosphorylation by cyclinA-cdk2 is believed to interfere with the DNA binding activity of E2F (Krek et al., 1994; 1995).
The physiological regulation of E2F activity imparted by these afferent signalling proteins can be subverted by viral oncoproteins, such as adenovirus E1a, human papilloma virus E7 and SV40 large T antigen, through their ability to release active E2F by sequestering pocket proteins and cyclin/cdk complexes (Bandara and La Thange, 1991; Chellappan et al., 1991; 1992; Morris et al., 1993). This property correlates with the ability of these viral oncoproteins to transform tissue culture cells, again implicating E2F as an important physiological target in virally-medicated oncogensis.
Considerable progress has been made in elucidating the composition of E2F. It is now known the E2F DNA binding activity defined in mammalian cells extracts is a generic activity caused by an array of DNA binding heterodimers made up from two distinct families of proteins, known as E2F and DP (La Thangue, 1994). Five members of the E2F family, from E2F-1 to E2F-5, have been isolated, each protein possessing preferential specificity for pocket proteins (Helin et al., 1992; Kaelin et al., 1992; Shan et al 1992; Ivey-Hoyle et al., 1993; Lees et al., 1993; Beijersbergen et al., 1994; Ginsberg et al., 1994; Buck et al., 1995; Hijmans et al., 1995; Sardet et al., 1995). For example, E2F-1 is regulated by pRb, and E2F-4 by p107 and p130 (Helin et al., 1993a; Flemington et al., 1993; Beijersbergen et al., 1994; 1995; Ginsberg et al., 1994; Vairo et al., 1995). Three members of the DP family are known (Girling et al., 1993; 1994; Ormondroyd et al., 1995; Wu et al., 1995; Zhang and Chellappan, 1995), DP-1 being a widespread and constitutive component of physiological E2F during cell cycle progression in some cell types (Girling et al., 1993; Bandara et al., 1994). Supporting their role as dominant regulators of the cell cycle, both E2F and DP proteins have been shown to possess proto-oncogenic activity (Johnson et al., 1994; Jooss et al., 1995).
Our previous characterisation of DP-3 indicated that it is a novel member of the DP family of proteins and that its RNA undergoes extensive alternative splicing (Ormondroyd et al., 1995). Processing events in the 5xe2x80x2 untranslated region and coding sequence of the RNA give rise to a rage of products present in both cell lines and tissues (Ormondroyd et al., 1995). A sequence of 16 amino acid resides within the N-terminal region of the DNA binding domain, known as the E region, is one such region subject to the alternative splicing of DP-3 RNA. Further, in the four DP-3 protein products which have been characterised, xcex1 and xcex4 constitute E- forms, whereas xcex2 and xcex3 are E- variants (Ormondroyd et al., 1995). although E-; extensive sequence conservation is apparent across the DP protein family, a comparison of the known DP protein sequences indicated that they fall into two categories, being either E- or for example, DP-1 is an E-variant.